Since its conception more than 25 years ago, the principle of particle counting and sizing invented by Wallace H. Coulter has resulted in numerous methods and apparatuses for the electronic counting, sizing and analysis of microscopic particles, which are scanned in a fluid suspension, as shown by the pioneer U.S. Pat. No. 2,656,508 to Coulter. In this prior art arrangement, a D.C. electric current flow is established between two vessels by suspending electrodes in the respective bodies of the suspension fluid. The only fluid connection between the two bodies is through an orifice; hence, an electric current flow and field are established in the orifice. The orifice and the resultant electric field in and around it constitute a sensing zone. As each particle passes through the sensing zone, for the duration of the passage, the impedance of the contents of the sensing zones will change, thereby modulating the current flow and electric field in the sensing zone, and hence causing the generation of a signal to be applied to a detector suitably arranged to respond to such change. (The mark "Coulter" is a registered trademark, Registration No. 995,825, of Coulter Electronics, Inc. of Hialeah, Florida.)
It has been proven that the change in impedance of the contents of the sensing zone as a particle passes through it is approximately proportional to the volume of the particle, where the cross-sectional area of the particle is substantially smaller than the cross-sectional area of the orifice, and the particle is smaller in diameter than the axial length of the orifice. Accordingly, numerous embodiments of commercial particle analyzers have been developed which measure signal amplitude output of an impedance sensing arrangement, for the purpose of measuring particle volume or size of the particles. Such an arrangement measures the electrical size of the particle which will be hereinafter referred to as "particle size" or "measured size".
It has also been proven that the particle's shape affects the measured size so that it does not correlate exactly with the actual or true volume of the particle. Generally, due to the hydrodynamic focusing in most apparatuses, elongated particles will be aligned with their elongated axis substantially parallel to the center axis of the orifice. With two equal volume particles, one being spherical and one being elongated, the spherical particle, while passing through the orifice, will have a greater cross section perpendicular to the current flow than the elongated particle. Hence, the spherical particle will distort the field in such a manner that it will give a greater measured size than the elongated particle, despite their equal volumes. To compensate for this, particles have been classified as to their shape by a term called "shape factor". For instance, if an extremely elongated particle is assigned a shape factor of 1.0, then the spherical particle of the same volume has a shape factor of 1.5. An apparatus using two sensing orifices capable of determining shape factor is shown in U.S. Pat. No. 3,793,587 to Thom et al. In this device, the length of one of the orifices has the same order of magnitude of the particle lengths or is smaller than the particle lengths. Consequently, with this orifice, an elongated particle causes a pulse, which after rising, remains at a maximum for a certain length of time and then falls. A spherical particle produces, in contrast, a pulse that falls immediately after reaching a maximum. In this patent it is suggested that the measured size can be corrected by dividing the impedance change for a particle by its shape factor. Due to complications of electric fields, these corrections leave much room for improvement.
Particle deformability, caused by hydrodynamic pressures as the particle proceeds through a sensing orifice, or particle shape can be very important, both as a factor affecting measured size and as a separate parameter for examining particles. First, the deformation or shape of the particles affects their shape factor, which in turn affects the measured size. Secondly, the deformed state of a biological cell depends not only upon the type of cell, but upon the age of the cell. For instance, mammalian erythrocytes have no nucleus. In contrast to leukocytes, the erythrocytes are easily deformable, due to their low inner viscosity. Also, within a given type of cell, the cell membrane becomes more rigid with age, therefore less deformable. It is contemplated that the pathological state of a cell will affect its deformability or natural shape.
In the commercial apparatus constructed in accordance with the heretofore mentioned U.S. Pat. No. 2,656,508, field excitation has been supplied by a direct current or low frequency source. As previously described, the electrical change caused by the passage of a particle through the electric field of small dimensions, excited by a direct or low frequency current, is approximately proportional to particle size. A direct current is considered to be of zero frequency in this application. However, the impedance sensing principle has been materially expanded to provide information concerning particles being studied, not limited only to characteristics due to the size of particles, but including characteristics due to the composition and nature of the material constituting the particles, as disclosed in U.S. Pat. No. 3,502,974 to Coulter et al. and U.S. Pat. No. 3,502,973 to Coulter et al. These prior art apparatuses generally have at least two current sources, both of which are applied to the sensing zone simultaneously, one having a radio frequency and the other being a "zero frequency" direct current or, alternatively, having a sufficiently low frequency that the reactive part of the particle impedance has a neglible effect on the response of the apparatus. One of the useful particle descriptors that can be obtained from this dual source arrangement is known in the art as the "opacity" of the particles. In a general sense, opacity measures the difference in size as measured at radio frequency as compared to size measured at low or zero frequency.
As is appreciated in the art of cytology, any new particle descriptor that can be measured is useful in identifying, analyzing and sorting particles. For example, cells have a membrane of very high resistivity which is in the range of a dielectric. However, the internal portion of the cell is fairly conductive, with different types of particles having varying internal resistivities. Also, it is contemplated that the pathological state of the cell will affect its internal resistivity. Consequently, it is desirable to measure this internal resistivity on a cell by cell basis.
U.S. Pat. No. 3,890,568 to Coulter et al. is of interest in that it discloses the electrical field configuration for an illustrative sensing orifice. Moreover, this patent teaches the measurement of particle length for the purpose of correcting inaccurate size measurements caused by elongated particles which exceeded the length of the sensing zone. However, the procedure disclosed therein is capable of accurately determining length of the particle only when the particle's length exceeds the effective scanning ambit, or to put it another way, the range within which the particle can be effectively sensed. This situation generally occurs only in instances when such particles as fibers are being sorted. With most particles, such as biological cells, the length of the orifice will be several times the length of the particles, even when the particles are stretched by the hydrodynamic forces involved.
The device disclosed in an article entitled "Fast Imaging in Flow: A Means of Combining Flow-Cytometry and Image Analysis", V. Kachel et al., THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, Vol. 27, No. 1, (1979), pp. 335-341, is of interest for disclosing a prior art scheme that is capable of subsequent examination of particle shape, after the particle has passed through a sensing orifice. In this device, an electronic unit associated with the sensing orifice, for a limited, preselected subpopulation of cells, triggers a flashlamp to project images of the selected cells on a film, for subsequent storage and examination. However, this arrangement has no capability of correlating particle shape with measured volume on a particle by particle basis. Shape information cannot be readily quantified in a rapid manner for computer processing, since its final form is nothing more than an image on the film. Moreover, flow speeds are limited to 5 meters per second with a maximum of only 150 pictures being taken per second, and then only for particles that are preselected. Typical flow speeds are 5 to 10 meters per second with a particle count rate of 1,000 to 5,000 particles per second.
An impedance sensing orifice has been used in combination with downstream light absorbance detection, scattered light detection and fluorescent light detection, as disclosed in U.S. Pat. No. 3,710,933 to Fulwyler et al. However, the types of optical measurements made downstream from the orifice do not provide the information required for the hereinafter described invention. Other prior art arrangements have simultaneously measured impedance and the above mentioned optical signals in optically clear flow cells. Although scattered light patterns are affected by particle shape, this effect is detectable and discernible only by extremely complex apparatus with marginal accuracy, since it is masked by scattered light created by reflected light, which is primarily dependent upon particle size, and refracted light, which is primarily dependent upon the particle's light transmission characteristics, namely absorption and refractive index, and size.
Various slit scanning techniques for analyzing particles with a narrow beam of light are known in the art, as illustrated by an article entitled "Imaging in Flow", D. B. Kay et al., published by THE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, Vol. 27, No. 1, (1979), pp. 329-334.